Raman spectroscopy is a technique which uses inelastic or Raman scattering of monochromatic light. Conventionally, the monochromatic light source is a laser in the visible or near infrared (“NIR”) range. The energy of the scattered photons is shifted up or down in response to interaction with vibrational modes or excitations in the illuminated material, varying the wavelength of the scattered photons. Accordingly, the spectra from the scattered light can provide information about the scattering material.
NIR Raman spectroscopy is known as a potential technique for characterisation and diagnosis of precancerous and cancerous cells and tissue in vivo in a number of organs. The technique is desirable as it can be non-invasive or minimally invasive, not requiring biopsies or the other removal of tissue. It is known to use NIR Raman spectroscopy in two wavelength ranges. The first is the so-called fingerprint (“FP”) range, with wave numbers from 800 to 1800 cm−1, owing to the wealth of highly specific bimolecular information, for example from protein, DNA and lipid contents, contained in this spectral region for tissue characterisation and diagnosis. The disadvantage of this wavelength range is, that when used with a commonly used 785 nm laser source, the strong tissue autofluorescence background signal can be generated. Further, where the probe uses optical fiber, a Raman signal is scattered from the fused silica in the optical fibers. In particular, where a charge-coupled device (“CCD”) is used to measure the scattered spectra, the autofluorescence signal can saturate the CCD and interfere with the detection of the inherently very weak Raman signals in this wavelength range.
Another problem with fiber-optic Raman spectroscopy as a technique is that of standardization of instruments. The fiber-optic Raman spectroscopy technique has mainly been limited to single systems and no attempts have been made to transfer into multi-centre clinical trials or routine medical diagnostics. This is mainly because Raman spectrometer instruments are generally dissimilar (i.e., optics, response function, alignment, throughput etc.) and in general produce very different Raman spectra. Further, fiber optic Raman probes have limited lifetimes and must be replaced or interchanged periodically. Unfortunately, Raman data acquired using different fiber optic probes cannot be compared, because each fiber optic probe has its own unique background as well as being associated with different transmissive spectral properties. The different transmissive characteristics significantly distort the spectral intensities making the tissue Raman spectra obtained with different fiber optic probes incomparable. As a consequence, multivariate diagnostic algorithms developed on a primary clinical platform cannot be applied to secondary clinical platforms. In particular, the quantitative measurement of tissue Raman intensity is one of the most challenging issues in fiber optic biomedical Raman applications. The instrument/fiber probe-independent intensity calibration and standardization is essential to the realization of global use of fiber optic Raman spectroscopy in biomedicine. For this reason, a multivariate statistical diagnostic model constructed using a ‘master’ probe cannot be applied to spectra measured with a ‘slave’ probe. In order for Raman technique to become a widespread tool for cancer screening on a global scale, there is a need to standardize both Raman spectrometers and fiber optic probes especially for biomedical applications. Most of the reported studies have focused on inter-Raman spectrometer standardization for measurements of simple chemical mixtures without fiber optic probes. In general Raman spectroscopy of simple chemical mixtures cannot be compared with the fiber optic Raman spectroscopy of heterogeneous biological tissue samples.
A further problem with standardizing results across instruments is that of spectral variation associated with the laser excitation power. Conventionally, Raman spectra are normalized which preserves the general spectrum shape, but this removes the absolute quantitative spectral characteristics. It has been known to attempt to monitor delivered laser power in fibre-optic Raman probes by, for example, embedding a diamond in the fibre tip or locating a polymer cap in the laser light path as a reference. However, these solutions are not satisfactory and may cause interferences in the required spectral regions.
A further problem in using optical spectroscopic techniques (including reflectance fluorescence and Raman) for in vivo diagnosis of cancer and precancerous conditions is that data analysis mostly been limited to post-processing and off-line algorithm development. This is true for endoscopic analysis because a large number of spectra collected during endoscopy are outliers. It would be useful to have a system that allows for real-time diagnosis for endoscopy.